1. Field of the Invention
This invention relates to antigens produced by the liver fluke organism Fasciola.
2. Brief Description of the Drawing
The accompanying drawing shows schematically the life cycle of Fasciola hepatica.
3. Description of Prior Art
Fasciola hepatica infections in cattle and sheep are reported to be responsible for losses due to poor growth and low milk yields valued in 1974 at (40 million per annum in the UK alone. It is also known that liver fluke increases susceptibility to salmonellosis in cattle. F. hepatica infections are also a serious problem in sheep, and increase susceptibility to "Black disease" caused by Clostridium oedematiens.
F. hepatica is a form of parasitic worm and has a complex life cycle involving more than one host which can more easily be understood from the accompanying drawing. The mature (adult) flukes reside in the bile ducts of a vertebrate host (cattle, sheep, etc.), from which eggs pass into the intestine and eventually onto pastures in faeces. After embryonation, miracidia are formed which hatch to infect certain species of snails (the second host). Asexual reproduction occurs in the snail and after some weeks the pre-infective form of fluke, known as cercariae, is released. A single miracidium typically gives rise to 200-600 cercariae. The cercariae anchor themselves to suitable substrates such as grass and secrete refractory coats or cysts. This process is called encystment. The encysted stage is known as a metacercaria and it is in this form that the parasite enters the vertebrate host, thereby infecting it. After ingestion by the vertebrate host, the juveniles emerge from their metacercarial cysts in the small intestine (a process known as excystment). The emergent free juveniles are hereinafter referred to as 0-day juveniles, to indicate the infective stage of the juveniles, at which they are ready to infect the animal. In this specification the "juvenile" stage means all stages from immediately before infection up to 4 days post infection (p.i.) in mice. (The length of the juvenile stage varies from one animal species to another). The juveniles migrate across the peritoneal cavity to the liver and thence to the bile duct, wherein they mature to adulthood.
Although there are many good chemotherapeutic agents available to combat liver flukes, the majority are ineffective against the earliest stages of a primary infection. Since primary infections often cause chronic, sometimes acute, disease, it would be advantageous to immunise the animals to protect against the earlier stages of primary infection. Dead adult flukes are ineffective, see K. B. Sinclair et al., Res. Vet. Sci. 16, 320-327 (1974). These observations have led to the hypothesis that a change in surface antigens occurs during the growth and development of F. hepatica. The juvenile and adult forms of fluke have a tegument, which includes a surface cytoplasmic layer connected to sub-muscular cellular regions by cytoplasmic tubes. The structure and cellular composition of the tegument changes with maturation of the fluke. The cytoplasmic layer has on its surface a plasma membrane which has on it a glycoproteinaceous surface layer which is known as the glycocalyx. The newly emerged 0-day stage juvenile flukes (NEJs) possess a glycocalyx specific to the juvenile stage, which is subsequently shed and exchanged for another type of glycocalyx before adulthood. The fluke replaces the shed layer after a short period by secretions from the tegument. It therefore changes the outermost surface which it presents to the vertebrate host and therefore the surface antigens which have stimulated an immune reaction in the host. It is thought that by this means the parasite evades immune attack, since by the time that the host has been stimulated to produce antibodies to the juvenile stage specific surface antigens presented, the fluke has changed its surface. In this way, a primary infection of fluke may evade the immune response for long enough to enter the liver parenchyma (a relatively protected and nutritious environment where the flukes of a primary infection are thought to avoid immune attack by turnover of the glycocalyx i.e. continuously sloughing off).
It will be evident from the above account that the formulation of a vaccine against F. hepatica infection is likely to be very difficult. An early paper supporting the above hypothesis is by Dr. C. E. Bennett, the present inventor, in Parasitology 77, 325-332 (1978). Antiserum against adult F. hepatica raised in rabbits reacted with the surface coat of formaldehydefixed adult flukes of both rat and mouse origin, as demonstrated by an indirect fluorescent antibody test (IFAT). A lack of reaction with live flukes indicated an active turnover of the surface antigen. Bennett also found that the adult antiserum did not react with fixed newly-excysted juveniles (NEJs), when they were very young viz. at 1 or 2 days post-infection (p.i.). At 5 days p.i. or older they did react. The existence of juvenile antigens was later demonstrated by the inventor, Dr. C. E. Bennett, with the valuable assistance of Mr. G. W. P. Joshua and Dr. D. L. Hughes, J. Parasitol, 68, 791-795 (1982). In this demonstration rabbits were injected with a crude preparation of antigens of metacercariae of F. hepatica, generating antibodies to a full range of somatic antigens. This mixture of antibodies was then "back-absorbed" with adult stage antigen (recovered from infected rats) and the antigen-antibody precipitate was removed. Thereby, all the antibodies to general somatic antigens present throughout maturation were precipitated, leaving an antiserum hypothetically containing anti-juvenile antibodies. This antiserum was tested for reaction with juvenile flukes of varying ages, using an IFAT. Freshly excysted 0-day juveniles gave a strong positive reaction. The degree of reaction fell with increasing age of the flukes under test, i.e. up to 4 days post infection in mice. This paper by Bennett et al. supports the postulate that there are juvenile-specific antigens, but, as the authors specifically say in their paper at page 794, right-hand column, does not indicate that juvenile-specific antigens are functional in stimulating immunity. Nor does it describe the preparation of antigens.
The literature is unclear on the issue of whether early or juvenile-specific antigens stimulate immunity. T. J. Hayes et al., J. Parasitol 58, 1103-1105 (1972) found that rats already infected with liver fluke were immune to reinfection by live metacercariae. This indicated that the immunological memory of the host, at least in rats, includes any juvenile antibody component that there might be. This was in contrast to J. C. Boray, Annals of Tropical Medicine and Parasitology 61439-450 (1967) who challenged liver fluke-free and infected sheep with live metacercariae and concluded that there was no appreciable difference between the two groups of sheep as judged by number of flukes excreted, clinical symptoms or pathology.
M. J. Howell et al., International Journal of Parasitology 9, 41-45 (1979) produced an antibody-antigen precipitate in vitro, by culturing the serum of liver fluke-infected rats with metacercariae. Vaccinations of rats with the precipitate in Freund's Complete Adjuvant (FCA) confirmed a significant degree of protection against an oral challenge with metacercariae in one experiment but no significant difference over the control in the other experiment. Subsequently Howell confirmed protection in Wistar rats, with modifications only to the route of immunisation His results showed significant levels of protection, 87% and 63% in separate experiments. In his first experiment 5 out of 6 rats vaccinated were completely protected showing no signs of liver damage or enlarged bile ducts. In his second experiment 2 out of 6 rats vaccinated similarly showed no signs of liver damage. See Journal of Parasitology 65, 817-819 (1979).
The above method of immunisation, involving use of an immune complex, was followed up in sheep, R. M. Sanderson and M. J. Howell, Res. Vet. Sci. 29. 255-259 (1980). Sheep were injected intramuscularly with a mixture of FCA and the complex obtained from in vitro culture of excysted metacercariae (in effect NEJs) and the serum of liver fluke-infected sheep. However, no protection was conferred on the sheep.
It is known from field observations that sheep and cattle do develop natural immunity to reinfection by liver fluke, see e.g. J. G. Ross, Journal of Helminthology 41, 393-399 (1967). There is experimental evidence of the role of the immune system, in that sheep can be made to develop immunity to liver fluke by the T-cell stimulant levamisole. Levamisole-treated sheep were infected with Cysticercus tenuicollis, challenged intra-ruminally with liver fluke metacercariae, and found to have a substantial level of resistance to liver fluke infection. See N. J. Campbell et al., Int. J. Parasitol. 7, 347-351 (1977) and J. K. Dineen et al., Int. J. Parasitol. 8, 173-176 (1978). G. B. B. Mitchell et al., Res. Vet. Sci. 30, 343-348 (1981) found that sheep first infected with nematodes (having no biological similarity to F. hepatica) and then treated with levamisole acquired immunity to F. hepatica. These authors speculated that an immunosuppressive component is involved in the pathogenesis of live fluke infection and that levamisole acts to correct the suppression.
The European patent Office Search Report RS 71916 GB on the priority UK application has cited four references, which will be briefly reviewed.
Biological Abstracts 75 72780 (1983) is an abstract of the Bennett et al. paper referred to above.
Chemical Abstracts 73 128965t (1970) abstracts R. Tailliez et al, Biologie Medicale 59, 183-287 (1970). The paper reviews immunological work on F. hepatica and reports the isolation and purification from adult F. hepatica of five immunologically active fractions in the fluke.
European patent Specification 11438A (Vaccines International Limited) describes a fascioliasis vaccine, especially for bovine administration, comprising irradiated metacercariae of Fasciola gigantica (which is the causative agent of fascioliasis in cattle in Africa). However, the previous attempted use of irradiated metacercariae for vaccination of sheep against F. hepatica was unsuccessful according to Biological Abstracts 66 71066 (1978) abstracting N. J. Campbell et al., Vet. Parasitol 4, 143-152 (1978).
PCT Application WO 83/00229 (The John Hopkins University) relates to diagnosis of fluke infections, and a method of passive vaccination which comprises administering monoclonal antibodies defined by their hybridoma cell lines. It is described in detail by reference to schistosomes, i.e. parasites of the genus Schistosoma, and is "premised on the discovery that the membrane of flukes in all growth phases, including eggs, contain two major glycoprotein molecules", referred to as fluke spine glycoproteins. It is suggested that F. hepatica and F. gigantica can be detected analogously.